RNA editing is a natural process through which eukaryotic cells alter the sequence of RNA molecules, often in a site-specific and precise way, thereby increasing the repertoire of genome encoded RNAs by several orders of magnitude. RNA editing enzymes have been described for eukaryotic species throughout the animal and plant kingdoms, and these processes play an important role in managing cellular homeostasis in metazoans from the simplest life forms, such as Caenorhabditis elegans, to humans. Examples of RNA editing are adenosine to inosine and cytidine to uridine conversions through enzymes called adenosine deaminase and cytidine deaminase, respectively. The most extensively studied RNA editing system is the adenosine deaminase enzyme. Adenosine deaminase is a multidomain protein, comprising a recognition domain and a catalytic domain. The recognition domain recognizes a specific dsRNA sequence and/or conformation, whereas the catalytic domain converts an adenosine into inosine in a nearby, more or less predefined, position in the target RNA, by deamination of the nucleobase. Inosine is read as guanine by the translational machinery of the cell, meaning that, if an edited adenosine is in a coding region of a mRNA or pre-mRNA, it can recode the protein sequence. A to I conversion may also occur in 5′ non-coding sequence of a target mRNA, creating new translational start sites upstream of the original start site, which gives rise to N-terminally extended proteins. In addition, A to I conversions may take place in splice elements in introns or exons in pre-mRNAs, thereby altering the pattern of splicing. Exons may be included or skipped, as a consequence of such RNA editing. The adenosine deaminases are part of the extensive family of enzymes called adenosine deaminases acting on RNA (ADAR), including human deaminases hADAR1, hADAR2 and hADAR3.
The use of oligonucleotides to edit a target RNA is known in the art, e.g. see Montiel-Gonzalez et al. (Proceedings of the National Academy of Sciences 2013 Nov. 5, 2013, vol. 110, no. 45, pp. 18285-18290). The authors described the targeted editing of a target RNA using a genetically engineered fusion protein, comprising an adenosine deaminase domain of the hADAR1 protein, fused to the so-called B-box binding domain of bacteriophage lambda protein N. The natural recognition domain of hADAR1 had been removed to eliminate the substrate recognition properties of the natural ADAR and replace it by the B-box recognition domain of lambda N-protein. The B-box is a short stretch of RNA of 17 nucleotides that is recognized by the N-protein B-box binding domain. The authors created an antisense oligonucleotide comprising a guide RNA part that is complementary to the target sequence for editing fused to a B-box portion for sequence specific recognition by the N-domain-deaminase fusion protein. The authors elegantly showed that the guide RNA oligonucleotide faithfully directed the adenosine deaminase fusion protein to the target site, resulting in gRNA-directed site-specific A to I editing of the target RNA.
A disadvantage of the proposed method is the need for a fusion protein consisting of the B-box binding domain of bacteriophage lambda N-protein, genetically fused to the adenosine deaminase domain of a truncated natural ADAR protein. This requires the target cells to be either transduced with the fusion protein, which is a major hurdle, or that the target cells are transfected with a nucleic acid construct encoding the engineered adenosine deaminase fusion protein for expression in the target cells. The latter constitutes no minor obstacle when editing is to be achieved in a multicellular organism, such as in therapy against human disease.
Vogel et al. (Angewandte Chemie. Int. Ed. 2014, 53, 6267-71) disclose editing of eCFP and Factor V Leiden coding RNAs using a benzylguanine substituted guideRNA and a genetically engineered fusion protein, comprising the adenosine deaminase domains of ADAR1 or 2 genetically fused to a SNAP-tag domain (an engineered O6-alkylguanine-DNA-alkyl transferase). Although the genetically engineered artificial deaminase fusion protein could be targeted to a desired editing site in the target RNAs in Hela cells in culture, using covalently linked guide RNA (through benzylguanine), this system suffers from similar drawbacks as the genetically engineered ADARs described above, in that it is not clear how to apply the system without having to genetically modify the ADAR first and subsequently transfect or transduct the cells harboring the target RNA, to provide the cells with this genetically engineered protein. Clearly, this system is not readily adaptable for use in humans, e.g. in a therapeutic setting.
Another editing technique which uses oligonucleotides is known as CRISPR/Cas9 system, but this editing complex acts on DNA. The latter method suffers from the same drawback as the engineered ADAR systems described above, as it requires co-delivery to the target cell of the CRISPR/Cas9 enzyme, or an expression construct encoding the same, together with the guide oligonucleotide.
Hence, there remains a need for new techniques which can utilise endogenous cellular pathways to edit endogenous nucleic acids in mammalian cells, even in whole organisms, without the problems associated with the methods of the prior art.